Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 315

thus ensuring the function of GFP as an autofluorescent protein. 7,8 The
protein folding and chromophore formation processes are likely to be
interdependent. 9 Indeed, the residues near the chromophore participate
in determining the photochemical properties of each variant of GFP. 10,11
In addition, unfolding of GFP, which radically dismantles the

-barrel and
repositions the amino acids surrounding the chromophore, completely
abolishes fluorescence, although the covalent chemical structure of the
central chromophore is maintained. 12
Spectral tuning of fluorescent proteins is achieved via substitution of the
amino acids composing the chromophore, which alters the extent of
p

b

-conjugation within the chromophore per se and also within the amino
acids surrounding the chromophore. The

b

-barrel protects the chromo-
phore from the environment and restricts its flexibility. Increasing evidence
regarding the relationship between the structure and the optical properties of
fluorescent proteins has led to the rational, direct manipulation of these
properties. To date, a large number of mutants of the original GFP, as well
as a range of fluorescent proteins originating from cnidarians other than
jellyfish, have therefore become available and together constitute a wide
palette of colors. Such proteins have been widely used to visualize protein
dynamics and environmental changes in living cells, although until now
their use has been restricted to basic research. For those interested in a more
complete survey, there are a number of excellent reviews that cover the
entire palette of fluorescent proteins currently available. 6,13-16
The ability of fluorescent proteins to emit visible light derives from the
posttranslational self-modification of three amino acids at positions 65-67
(Ser-Tyr-Gly in A. victoria GFP and Thr-Tyr-Gly in enhanced GFP)
( Fig. 8.1B and Table 8.1 ), which results in chromophore formation. 3
Among these three chromophore-forming amino acids, only the glycine
residue located at position 67 is absolutely conserved within all fluorescent
proteins ( Table 8.1 ). Although the tyrosine residue at position 66 is con-
served in all naturally occurring GFP-like proteins ( Table 8.1 ), it can be
substituted with any aromatic amino acid. This residue is believed to provide
the proper oxidative chemistry during chromophore maturation and
prevent undesirable side reactions,

such as hydrolysis and backbone

fragmentation ( Fig. 8.1B ). 17,18
Substitution of the tyrosine residue with an aromatic amino acid results
in chemically distinct chromophores that emit light in the blue or cyan
range 3 ; for example, histidine is substituted in EBFP (enhanced blue fluo-
rescent protein) and tryptophan in ECFP (enhanced cyan fluorescent